Abstract

Vibrio parahaemolyticus is a halophilic bacterium associated with seafood gastroenteritis. An unusual strain of Kanagawa-positive urease producing Vibrio parahaemolyticus O1 : K1 was isolated from the environment and identified. A polymerase chain reaction assay revealed that this strain harbored both the tdh and trh genes. The urease from this strain was studied. Maximum urease production was induced in LB medium containing 0.2% urea, 0.5% glucose, 2% NaCl and pH 5.5 with 6 h of cultivation at 37 C under aeration. Purification of urease was achieved by the process of whole cell lysate, 65% ammonium sulphate precipitation, DEAE-cellulose ion exchange column chromatography, Sepharose CL-6B gel filtration and oxirane activated Sepharose 6B-urea affinity chromatography with 203 fold purification and 2.2% yield. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of the subunits with a molecular weight of 85 kDa, 59 kDa, 41 kDa and the molecular weight for the native enzyme by nondenaturing PAGE and gel filtration chromatography was 255 kDa. The purified urease was stable at pH 7.5 and the optimal pH in HEPES buffer was 8.0. The enzyme was stable at 60 C for 2 h with a residual activity of 32%. The addition of 10 uM of NiCl_2 maintained stability for 30 min. The Km value of the purified enzyme was 35.6 mM in urea substrate. The TD_50 (median toxic dose) of the purified urease was 2.5 ug/ml on human leukemia cells.

• Keywords: urease; Vibrio parahaemolyticus